KR20160025023A - Solvent production - Google Patents

Abstract

The present invention relates to a process and system for producing a solvent using single-phase solvent-produced Clostridia in a single stage process. More specifically, the processes and systems relate to the use of culture vessels for culturing single-phase solvent-produced Clostridia, wherein the cell growth of clostridium is monitored by the addition of controlled volumes of culture medium and / or removal of control volumes of solvent Is optimized. Such processes and systems can be specifically used to produce solvents such as butanol, acetone, ethanol, and the like.

Description

Solvent Production {SOLVENT PRODUCTION}

The present invention relates to a process and system for producing a solvent using a monophasic solventogenic clostridia in a single stage process. More specifically, the processes and systems relate to the use of culture vessels for culturing single-phase solvent-produced Clostridia, wherein the cell growth of clostridium is monitored by the addition of controlled volumes of culture medium and / or removal of control volumes of solvent Is optimized. Such processes and systems can be specifically used to produce solvents such as butanol, acetone, ethanol, and the like.

The butanol fermentation process utilizes renewable bio-based feedstocks and is often referred to as acetone, butanol, and ethanol (ABE) fermentation along with the main chemical products. Fermentation was first commercialized in England in 1916 and spread throughout the world during World War I, mainly producing acetone for munitions and producing butanol for paint lacquers. The process lost its popularity in the United States and Britain in the 1950s as it competed in cost with oil-based equivalents, but continued to be popular in China, Russia and South Africa until the 1980s. Today, ABE fermentation is ready to be re-commercialized due to high oil prices, concerns about oil supply, and environmental concerns about greenhouse gas (GHG) emissions. The fermentation pathway has the potential to replace petroleum-based butanol, acetone and hydrogen with cheaper, more sustainable, environmentally friendly chemicals. In fact, global investment in bio-butanol has become more active by investing in new plants in China. To date, more than two million dollars have been invested to achieve a solvent capacity of 0.3Mt / year in an installed plant and to expand to 1Mt / year.

Traditional batch processes for the fermentation of molasses and / or starch to produce ABE have been conducted for decades (Jones, D. T. and Woods, D. R. (1986) Microbiol. Rev. 50: 484-524). Typically batch fermentation produces about 18 g / L of solvent for 72 hours. Fermentation takes considerably longer because it occurs in two separate stages (ideal: biphasic), where the first stage is the growth stage in which the acid is produced and the pH is lowered, and the second stage is the rejuvenation of the acids in the solvent Is a survival stage. Cells also prepare for sporulation. The switch of metabolism is initiated by the drop in acid concentration and / or pH of the fermentation broth. The final solvent titration is significantly lower than that of the yeast ethanol fermentation, thus resulting in lower volume productivity (18/72 equals approximately 0.25 g solvent / L / year). Solvent titration concentrations are limited by a fairly small amount of sugar that can be fermented. Low solvent productivity is a major drawback of traditional batch processes and many attempts have been made to overcome these limitations. Have been developed with variations of batch culture processes that include batch-feed processes that attempt to increase the solvent titration concentration or reduce the fermentation time, but have been largely unsuccessful.

Several single-stage continuous processes (such as those described in Jones and Woods, supra), which attempt to provide a continuous flow of feed medium to achieve a growth rate close to the maximum growth rate for several weeks due to the low productivity achieved in the batch fermentation process (1986)) have been proposed, thereby improving the solvent production rate and reducing downtime. In practice, however, this is difficult to achieve with ideal solvent-producing Clostridia because solvent production is not directly linked to growth, and the culture is washed at a significantly lower dilution rate. Methods that use cell-recycle and / or immobilization to maintain high cell concentration in the reactor have been proven on a laboratory scale, but are difficult to implement on a commercial scale (e.g. Qureshi & Maddox (1987), Enz. Microb. Technol Maddox (1989) Gen. Eng. Rev., 7, 189-220; Maddox et al. (1993) In: The clostridia and biotechnology, eds DR Woods, Butterworth-Heinemann , Boston, 343-369, Gapes et al., (1996) Appl. Env Microbiol., 62: 3210-3219).

Although other continuous configurations based on 2- or multi-stage vessels have been proposed to isolate and control abnormal fermentation, none have been successful due to difficulties in controlling the flow rate and corresponding dilution rate to maximize solvent productivity ). Mixed cell populations of acid-producing and solvent-producing cells rapidly accumulate in the first and second stages, causing oscillations in growth and solvent formation. If the flow rate is not controlled, for free cell suspension, this usually causes the culture to be washed if the growth slows at a slow flow rate or proceeds in a less optimal manner. Also, cultures tend to rapidly lose their ability to produce solvents (Woolley and Norris (1990) J. Appl. Bacteriol. 69: 718-728; Jones and Woods, (1986) supra; Kashket and Cao 1995), FEMS Microbiol. Rev., 17, 307-315; Afschar (1990) DE 3905624 A1). Afschar (1990) proposed a two-stage molasses fermentation process for the production of butanol and acetone, which features a chemostat with substrate limitations in the first stage to produce cells. A two-stage continuous culture for Clostridia was also proposed by Mutschlechner et al. (J. Mol. Microbiol. Biotechnol. (2000) 2 (1): 101-105). In this process, the system is designed to mimic the two steps of batch culture growth by transferring cells from the " acidic breakpoint " to the second stage using a first stage that grows cells as rapidly as possible. The second vessel is large enough to provide sufficient residence time to complete the solvent production. In all of these examples, the flow rates to the first and second stages were kept constant and were not adjusted in response to any growth related signals such as pH changes or cell density.

Two-stage continuous cultures have also been described with respect to immobilized biomass or cell-keeping (see, for example, Maddox et al. (1993) Gapes et al. (1996), supra). These authors describe a method of immobilization that uses a fixed dilution rate and keeps the microbe in the reactor so that it is not washed. These cultures can proceed at fast dilution rates and productivity, but the final solvent concentration tends to be very low for cost effective recovery. Further, in both examples, the solvent proper concentration and productivity oscillated widely. The main disadvantages of immobilization are the difficulty with cost and constant ratio. Operation over extended periods of time and / or use of feedstock with particulates is a problem due to blocking and contamination of the support matrix. In addition, these systems are prone to contamination and difficult to maintain sterilization.

In China, a sequential fermentation process has also been commercialized, in which a continuous or sequential batch process is used in which eight fermenters are connected together. The first two vessels (vessel 1 and vessel 2) are biomass generators, and fresh cultures are continuously re-seeded every 24 hours (via conventional seed trains). Once the biomass generator has been seeded, the substrate (feedstock) is fed continuously and, when filled, the liquid flows in the forward direction towards the vessels 3, 4 (working in parallel). These two vessels then feed the remainder of the fermentation train consisting of a series of sequential connected vessels (typically four) providing eighty fermentation vessels with a total process residence time of 72 hours. This complex process is designed for abnormalities (and limitations) associated with C. acetobutylicum, which generally require continuous reseeding to avoid microbial degradation (due to loss of the solvent plasmid). This process is manually controlled in a very small range to quickly respond to the process fluctuation. China's continuous process is described by Ni & Sun (2009), Appl. Microbiol. Biotechnol., 83, 415-423.

To date, fermentation process technology has been developed for ideal solvents for producing Clostridia. Metabolism is characterized by rapid growth and acid formation and subsequent transitions in which growth slows, mountains recommence, and solvent production begins. As a result, the fermentation time is considerably long.

However, there is a need to provide a process that provides continuous or extended solvent production for an initial onset of solvent, a shorter fermentation time, and a period of time greater than 50 hours if supplied continuously.

Accordingly, one object of the present invention is to provide a batch fed or continuous feed fermentation process that can optionally incorporate solvent removal with cell and water recycle operations and maintain high solvent productivity over a period of more than 50 hours. The inventors have discovered that such integrated systems can be used to convert high concentrations of sugar or other carbon feeds to solvents (acetone, butanol, and ethanol) at high yields over extended periods of time while minimizing substrate inhibition Respectively.

The present invention is based on the surprising discovery that some solvent-produced Clostridia is not substantially abnormal and is a single phase. For batch fermentation, single phase solvent production is characterized by simultaneous growth and solvent production with no obvious changes in production metabolism or switch from acid to solvent during the main growth phase. Indeed, acid and solvent are produced simultaneously, and the acid does not tend to accumulate in the culture medium. In such a clostridia, there is no need to wait for the second step before the solvent is produced. For such clostridia, growth and solvent production may occur in a single vessel. Solvent production can be controlled by monitoring cell growth or growth-related characteristics (e.g., gas production, sugar utilization, or acid production) and optimizing conditions for cell growth, A proper concentration can be obtained. This approach is in contrast to previous approaches in which the culture conditions are optimized for solvent production in the second step, which is growth in the acid production step or solvent production.

Batch fermentations for single and extra solvent-producing Clostridia are shown in FIGS. 2 and 3. FIG.

Thus, in one embodiment, the present invention provides a single stage process for solvent production,

(a) culturing single-phase solvent-producing clostridia cells in a liquid culture medium in a culture vessel,

(b) monitoring cell growth of the single-phase solvent-produced Clostridia cells in the culture vessel;

(c) continuously or semi-continuously introducing a new culture medium and / or nutrients into the culture vessel,

(d) continuously or semicontinuously removing the stream or part of the liquid culture medium containing the solvent (s) from the culture vessel and delivering the liquid culture medium to a solvent remover, and

(e) maintaining or increasing the cell density in the culture vessel,

The cell growth of the culture vessel is carried out,

(i) the amount or rate of new culture medium or nutrient to be introduced into the culture vessel and / or

(ii) by controlling the amount or rate of the liquid culture medium comprising the solvent (s) removed from the culture vessel.

Preferably, the single phase clostridium is

i) naturally mark simultaneous growth and solvent production during the main growth phase of batch fermentation,

ii) chemically modified to produce a solvent during growth, or

iii) genetically modified to produce a solvent during growth.

Preferably, clostridium is single-phase C. saccharop- butyl acetonitrile.

Most preferably, clostridium is a single phase clostridial saccharoprotein acetonucleic N1 strain, for example N1-4 (HMT) or N1-504.

Preferably, step (b)

(i) monitoring the production of one or more gases (e.g., hydrogen and / or CO ₂ );

(ii) monitoring the production of one or more acids;

(iii) monitoring the pH of the culture medium;

(iv) monitoring the usage per party;

(v) monitoring the cell density; And

(vi) monitoring the production of one or more solvents

Lt; RTI ID = 0.0 > and / or < / RTI >

Advantageously, step (d) comprises continuously or semicontinuously removing a stream or part of the liquid culture medium from the culture vessel and delivering the stream / portion to a solvent remover through a cell separator,

(i) the cells are removed from the liquid culture medium in a cell separator, the cells are returned to the culture vessel (optionally through a cell seeder)

(ii) the solvent is removed from the liquid culture medium in a solvent remover, and the remaining liquid culture medium is returned to the culture vessel.

Preferably, the cell density,

(i) recycling the cells removed from the liquid culture medium containing the solvent (s) back to the culture vessel, and / or

(ii) continuously or semicontinuously feeding cells from the cell seeder into the culture vessel, and / or

(iii) fixing some or all of the cells in a culture container,

Maintained or increased in the culture vessel.

The process of the present invention can be carried out in any suitable manner. For example, the process may be implemented as a batch-feed process or as any form of continuous process or perfusion process, or as a mixture of these types of processes.

In some embodiments, the process is performed in batch-feed mode. In this embodiment, the microorganism is cultured under the desired growth conditions of the batch mode for a suitable period of time, for example, about 20 hours, until about half of the sugar (e.g. 25-30 g / L) is consumed. Microbial cells proliferate to produce both acid and solvent. The initial volume of the first batch stage of the process should preferably be about 70% (e.g., 65% to 75%) of the total workload of the vessel, and the growth and good solvent yield (For example, 55 to 65 g / L) and nutrients to maintain the volume of the fermentation vessel in the fermentation vessel. The fermenter must have a volume equivalent to about 30% (for example, 25% to 35% At a rate designed to last for a finite amount of time (e.g., 10 to 75 hours). The microorganisms can be grown under conditions suitable for optimal growth and solvent production (e. G. Temperature, pH, redox).

The process of the present invention is preferably carried out under continuous culture conditions. As used herein, the term "continuous culture conditions" refers to a condition in which the feed (including nutrients) in steady state conditions (defined by high water absorption and solvent productivity) for extended periods of time Quot; refers to a process by which the culture of microorganisms in a culture vessel can be maintained in a continuous or substantially continuous flow of the culture medium. In this situation, some bleed may be required to maintain a constant volume of the culture vessel.

Solvent-producing Clostridia is a bacteria capable of producing a solvent such as acetone, butanol and ethanol. Typically, the acids are first produced during the fermentation and then re-assimilated into the solvents.

The process of the present invention is a single stage process. As used herein, the term "single stage" means that both the acid (s) and solvent (s) are produced together in the same culture vessel. This may be in contrast to a two-step process in which the acids are produced in the first growth stage and the solvents are subsequently produced in the second stage and are typically carried out in two culture vessels.

Most solvent-produced clostridia (e. G. Clostridium < RTI ID = 0.0 > Acetobutylicum), fermentation is abnormal, the first phase is characterized by cell growth, acid production, and a drop in the culture pH, and in the second phase, the acids are converted to solvents and the culture pH is increased. The switch of metabolism is initiated by the acid concentration and / or low culture pH value of the fermentation broth.

However, the present invention relates to single-phase Clostridia. As used herein, the term "single-phase clostridia" means that clostridia does not have a separate acid and solvent production phase. Single phase solvent production is characterized by simultaneous growth and solvent production with no obvious switch or change in metabolism. The acids tend not to accumulate in the culture medium.

Preferably, the microorganism is a single phase solvent producing clostridium.

Preferably, the microorganism is an ideal clostridium that has been converted to display single phase fermentation by chemical mutagenesis or by specific genetic modification or a combination thereof.

More preferably, the microorganism naturally expresses single-phase metabolism without requiring modification.

More preferably, the clostridium is a single phase clostridium ≪ / RTI >

Most preferably, clostridium is a single phase C. < RTI ID = 0.0 > N1 strain, such as N1-4 (HMT) or N1-504, or mutants or derivatives thereof. Such variants / derivatives will also be single-phase solvent-produced clostridia. Preferably, the variant / derivative produces the same or more acetone, ethanol and / or butanol compared to Clostridia, which is induced under equivalent conditions.

Such variants can be produced, for example, by random mutagenesis or by recombinant methods. Recombinant methods include insertion of gene inactivity through the use of Type II introns such as Targetron (Sigma) and Clostron (e.g. WO 2007/148091), and allelic coupling (ACE, e.g., WO 2009/101400) And integration of new genes through use. In addition, strains of acetate-resistant (Yang, S., Land, ML, Klingeman, DM, Pelletier, DA, Lu, TY, Martin, SL, Guo, HB, Smith, JC, . Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae . Proc Natl Acad Sci US A., 107 (23), 10395-400. 2010.), producing more ethanol-resistant strains (Shao, X., Raman, B., Zhu, M., Mielenz, JR, Brown, SD, Guss, AM, & Lynd, LR Selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum . Appl. Microbiol Biotechnol. 92 (3), 641-52. 2011. Also: Brown, SD, Guss, AM, Karpinets, TV, Parks, JM, Smolin, Smith, JC, Keller, M., Raman, B., Shao, X., M., Yang, S., Land, ML, Klingeman, DM, Bhandiwad, A., Rodriquez, M.J. , & Lynd, LR (2011). Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum . Proc Natl Acad Sci US A., 108 (33), 13752-7 2011.) (Annous, BA, & Blaschek, HP (1991). Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity. Appl. Environ. Microbiol., 57 (9), 2544-8. 1991.) Random mutagenesis techniques as already proven can also be used.

These mutants can be identified by looking for variants that produce the same or more acetone, ethanol, and / or butanol compared to clostridium, which are selected by small scale fermentation, i.e., maintaining single-phase behavior and inducing under equivalent conditions, It can be tested.

C. saccharide butyl hydroperoxide acetoxy T. glutamicum strain N1-4 (HMT) can be obtained from the deposited ATCC No. 27 021. C. Saccharomyces butyl hydroperoxide acetoxy T. glutamicum strain N1-504 can be obtained from the ATCC deposition number 27022.

In some embodiments of the present invention, Clostridium is not C. saccharophyta acetonuccum strain N1-4. C. Saccharomyces butyl hydroperoxide acetoxy T. glutamicum strain N1-4 was available in the past as the deposition number ATCC 13 564. However, this deposition is no longer available.

The term "acid-production" or "acid production" refers to the conversion of a sugar and / or starch-based substrate into RCOOH (where R is as defined below), for example, acetate and / or butyrate Indicates the ability of clostridium.

The term "solvent-produced" or "solvent generation" refers to the ability of clostridium to convert a substrate based on sugar and / or starch to a solvent, preferably one or more of acetone, ethanol and / Point.

As used herein, the term "solvent" or "solvents " refers to organic solvents of low boiling point or azeotropic liquids thereof, which can be produced by solvent-producing microorganisms in a liquid fermentation medium. Examples of such solvents are alcohols of the formula R-OH, wherein R is an aliphatic C1-C8 alkyl group or an aliphatic C2-C8 alkenyl group. The R group may be branched or linear. Preferably, the R group is linear. The R group can be saturated or unsaturated. Preferably, the R group is saturated.

Preferred examples of the alcohol of the formula R-OH include alcohols such as methanol, ethanol, 2-methyl-propan-1-ol 1,3-propanediol, 1-butanol, Hexanol, heptanol, and octanol. Additional examples of the solvent has a formula R-CO containing acetone _{((CH 3) 2 CO)} .

Preferably, the solvents include ABE solvents, i.e., acetone, 1-butanol and ethanol. Most preferably, the solvents comprise 1-butanol or substantially 1-butanol.

Clostridium may be an aerobic or anaerobic microorganism. Preferably, clostridium is anoxic or oxygen-resistant clostridium. Most preferably, clostridium is oxygen-resistant clostridium.

In some embodiments, the culture vessel may be inoculated without oxygen or with no specific instructions to avoid anaerobic purging. In addition, the culture vessel can be operated with air (of normal atmospheric composition) in the head space above the culture medium.

In some embodiments of the present invention, the process is performed under non-anoxic conditions.

The acid- and solvent-producing Clostridia used in the culture vessel may be from a single strain or from a co-culture, preferably from a single strain.

In some embodiments, Clostridia is acid resistant. Clostridia is preferably able to withstand high concentrations of COOH. In this regard, high concentrations of COOH can mean up to 15 g / L acetic acid and / or up to 10 g / L lactic acid and / or up to 6 g / L formic acid.

In some embodiments, some or all of the clostridia cells can be immobilized in a culture vessel. In other embodiments, the clostridial cells are not fixed or are free to be suspended.

The culture vessel may be any type of culture vessel suitable for culturing Clostridium cells in the process of the present invention. A preferred type of culture vessel comprises a conventional stirred bioreactor.

As used herein, the term "culture vessel" includes two or more culture vessels which may or may not be fluidly connected. Any number of culture vessels may be connected in parallel or in series.

However, in view of the present invention, both the acid (s) and solvent (s) are produced together (i. E., As a mixture) in a single culture vessel. One or more acids are not converted into one or more solvents in the second culture vessel after being produced in the first culture vessel.

Suitable culture media are known in the art. A suitable culture medium is selected according to the clostridium used. Generally, the culture medium is a liquid medium.

In embodiments of the present invention involving a batch feed process, fermentation proceeds in batch mode for about 20 hours with the reactor volume filled to about 70%. The medium should contain sufficient sugars and nutrients to allow the microorganisms to grow optimally and produce acids and some solvent (s).

In other embodiments, only about 10% of the reactor working volume can be initially filled and inoculated. About 60% of the remaining can be filled during cell growth.

The medium for the batch-feeding process may be a nutrient feed containing a sugar and / or nutrient concentration higher than that present in the culture vessel during the batch stage. Typical concentrations of sugars in the feed may vary from 250 g · L ^-1 to 450 g · L ^-1 . This may be supplied at a constant flow rate or may be controlled by a feedback system based on cell growth (or related features) or sugar or solvent levels in the culture vessel. In other embodiments, the addition of supplemental nutrient media may be initiated using an independent trigger rather than a trigger of solvent extraction, which may include, for example, when the sugar concentration is lowered between defined levels (e. / L) or a slowing of the growth rate is detected.

The feed rate of the additional sugar solution should preferably be kept in balance with the rate of absorption of the culture. This can be, for example, about 2 to 5 g · L ^-1 _· h ^-1 through the feeding stage to maintain a stable sugar concentration (eg, in the range of 5-30 g · L ^-1 ) in a culture vessel .

Clostridia in the culture vessel is maintained under conditions suitable for acid and solvent production. The conditions will include providing a nutrient medium comprising a suitable carbon source, for example, a carbohydrate that can be assimilated.

Examples of carbohydrates that can be assimilated are sugars such as C5 and C6 monomers, dimers per C5 and C6, sugar oligomers and sugar polymers. Preferred sugars are arabinose, xylose, mannose, fructose, glucose, galactose, sucrose, lactose, maltose, and cellobiose. Preferred polymers are starch, xylan, pectin, fructan, cellulose, and mannitol. Another suitable carbon source is glycerin.

Preferably, the sugar is a hydrolyzate derived from lignocellulosic feedstocks such as agricultural residues (corn & sugar), woody residues, energy crops or municipal wastes.

Carbohydrates may also be glucose-based polysaccharides, for example, starch or starch based materials. Most preferably, the carbohydrate is a starch or starch based material, such as corn, corn starch, corn mash, potato, potato starch, potato mash, potato peeling, potato chips, cassava, cassava starch, cassava chips, Starch, dextrin or "soluble starch ", such as those sold by Fisher / Sigma.

In some embodiments, the present invention may use a sugar or other carbon feed of greater than 200 g / L, with the substrate or solvent being minimally inhibited while maintaining the yield of the product in excess of 30% on a carbon feed basis.

The pH of the culture vessel can be controlled from the separate alkali feed or through the automated addition of alkali from the addition of feed medium with a lower pH (typically 1-2 pH units) than the culture vessel.

The pH of the culture container is preferably pH 5.5 to 7.0, more preferably pH 5.5 to 6.5.

In addition, the pH can be controlled by pH auxostat. Preferably, the pH oxostat has a separate alkali feeder and the pump is connected to a pump that controls the medium feeder. The cell density of the culture vessel can be controlled by changing the ratio or relative speed of the medium-feeding pump and the alkali-feed.

The temperature is chosen as the temperature at which the microorganism is best grown. For example, for mesophilic Clostridia, the temperature is preferably between 30 ° C and 37 ° C, more preferably between 31 ° C and 33 ° C, for example, about 32 ° C.

In step (b), the growth of single-phase solvent-producing Clostridia cells in the culture vessel is monitored. This is done to obtain information that can be used to maintain a good growth rate and high cell density in the culture vessel by the addition of new culture medium or nutrients and / or removal of the solvent.

Monitoring of the cell growth of Clostridia in the culture vessel may be done, but actual monitoring may or may not be performed in the culture vessel, for example monitoring can be done externally or against the sample removed from the culture vessel have.

Preferably, step (b)

(i) monitoring the production of one or more gases (e.g., hydrogen and / or CO ₂ );

(ii) monitoring the production of one or more acids;

(iii) monitoring the pH of the culture medium;

(iv) monitoring the usage per party;

(v) monitoring the cell density; And

(vi) monitoring the production of one or more solvents

Lt; RTI ID = 0.0 > and / or < / RTI >

During the process of the present invention, one or more gases can be produced by Clostridia.

These gases may include hydrogen and / or carbon dioxide.

The total amount of gas can be monitored using an anemometer, and individual gases can be quantified using a specific gas analyzer.

The ratio of gases can be monitored and used to control growth and solvent production by controlling the growth medium, the addition of nutrients, and / or the removal rate of the solvent.

Clostridia converts the substrate based on sugar and / or the whole to an acid (e.g., RCOOH, where R is as defined above). Exemplary acids include acetate and / or butyrate. The acid concentration can be monitored by gas chromatography (GC).

The production of these acids can also be monitored by changes in the culture pH. In this case, the monitoring device may be a pH meter.

The cell density can be monitored by optical means for determining the optical density (OD) at, for example, 600 nm.

In other embodiments, changes in cell density can be linked to changes in the rate of removal of the culture medium, including new solvent / nutrient feed rates or solvent (s), by using, for example, a turbidostat .

The production of one or more solvents (e.g., ethanol, butanol, acetone) can be monitored by gas chromatography (GC) and / or high performance liquid chromatography (HPLC).

Preferably, the butanol concentration of the culture vessel should not exceed 10 g butanol L < ^{-1 &} gt ;.

The use of one or more sugars (e. G., Glucose, xylose, fructose, arabinose, sucrose, cellobiose, or others suitable according to the feedstock) can be monitored by HPLC.

Preferably, the sugar concentration of the culture vessel should not exceed .l ^-1 per 30 g.

(d) comprises continuously or semicontinuously removing a stream or part of the liquid culture medium comprising solvent (s) from the culture vessel and delivering said liquid culture medium to a solvent remover. In the solvent remover, the at least one solvent is preferably isolated from the liquid culture medium and optionally isolated.

Advantageously, step (d) comprises continuously or semicontinuously removing a stream or part of the liquid culture medium from the culture vessel and delivering the stream / part to the solvent remover via a cell separator,

(i) the cells are removed from the liquid culture medium in a cell separator, the cells are returned to the culture vessel (optionally through a cell cider)

(ii) the solvent is removed from the liquid culture medium in a solvent remover, and the remaining liquid culture medium is returned to the culture vessel.

The liquid culture medium from the culture vessel can be delivered directly to the cell separator or indirectly.

The remaining liquid culture medium can, for example, be delivered directly or indirectly to the culture vessel and can, for example, be temporarily stored in a reservoir.

To reduce the loss of cells from the culture vessel, the cell separator may be located upstream of the solvent remover. In a cell separator, all or a substantial portion of the cells are removed from the stream or part of the liquid culture medium delivered through the cell separator. For example, the cell separator can remove at least 50%, preferably at least 70%, most preferably at least 80% or at least 90% of the cells from the liquid culture medium delivered through the cell separator.

Examples of cell separators are hollow fiber separators, membrane separators and centrifuges.

After separation from the liquid culture medium, the cells are returned to the culture vessel (optionally with some liquid culture medium) via an optional cell seeder.

The remaining liquid is then transferred to a solvent remover.

In step (ii) of step (d), the solvent is removed from the liquid culture medium by the solvent remover and the remaining liquid culture medium (i.e., the liquid culture medium from which some of the solvent or all of the solvent has been removed) is returned to the culture vessel.

The liquid culture medium to be removed from the culture vessel is enriched in at least one solvent produced by the solvent-produced Clostridia.

Preferably, the liquid culture medium is removed from the culture vessel once the solvent (s) produced in the culture vessel has reached a defined concentration point (e.g., 8 to 10 g of butanol L ^-1 liquid culture medium) Start.

The liquid culture medium may preferably be delivered to the solvent remover at a rate that keeps the solvent concentration of the liquid culture medium below the desired solvent concentration point. This concentration point may, for example, be a toxicity threshold for the particular solvent-produced clostridium used.

In general, the solvent (s) may be added to one or more of liquid-liquid extraction, ion-liquid extraction, gas stripping, vacuum evaporation, atmospheric or vacuum distillation, dialysis evaporation, ion exchange absorption, counter current solvent extraction and / (Fermentation broth). ≪ / RTI > Alternatively, for example, a hydrophobic membrane may be used with an air flux or an inert gas carrier or vacuum (dialysis evaporation) to aid separation (preferably in a continuous process).

Preferably, the solvent (s) is recovered from the liquid culture medium by atmospheric or vacuum distillation, dialysis evaporation, liquid-liquid extraction and / or gas stripping.

Preferably, the solvent extraction is carried out continuously.

In some embodiments of the invention, the solvent, for a period of time for a period in excess of 60 times out of at least 0.8g · L ^-1 · h ^-1 or 100 hours at a rate of 0.6g · L ^-1 · h ^{- 1} < / RTI > speed.

In some more preferred embodiments, the solvent extraction is by continuous atmospheric or vacuum distillation.

Once the solvents are removed from the liquid culture medium, some or all of the remaining liquid culture medium is returned to the culture vessel. This maximizes the use of nutrients from the liquid culture medium and minimizes water loss.

The present invention also relates to a solvent obtained by the process of the present invention.

In step (e), the cell density is preferably,

(i) recycling the cells removed from the liquid culture medium containing the solvent (s) back to the culture vessel;

(ii) continuously or semicontinuously feeding the cells from the cell cider to a culture vessel; And

(iii) fixing some or all of the cells in the culture container

Lt; RTI ID = 0.0 > and / or < / RTI >

In step (ii) of step (e), the cells are continuously or semi-continuously supplied from the cell cider to the culture container.

In the simplest version of the fermentation process, a single batch seed of 5-10% v / v can be used to inoculate the fermentation at the start of fermentation, but 5-10% in various time intervals during fermentation to maintain high cell density and solvent productivity. It is also possible to use multiple batch inoculations of v / v. Alternatively, continuous cultivation can be used to continuously feed new cells to the production fermenter. A preferred embodiment is a pH-oxostat which is fed from the bottom of the distillate's motionless and self-regulated using a set pH value to control the feed and / or the addition of fresh nutrients and sugars.

In step (e) of step (e), some or all of the clostridial cells may be fixed in a culture vessel.

Cells can be immobilized by active immobilization techniques whereby free suspension cells can be immobilized by covalent attachment to the surface, crosslinking of the cells to the surface, capture in the gel or membrane restraint, or electrostatic interaction By passive immobilization utilizing the natural tendency of the cells to adhere to the porous solid surface, or by the ability to form a film or agglomerate within the support material.

Preferred methods for immobilizing clostridia in the present application include,

(i) gel matrices or synthetic gels formed from naturally occurring polymers (e.g., alginic acid, kappa-carrageenin, collagen, gelatin, cellulose, etc.) (including polyacrylamides, polymethacrylamides, photocrosslinkable resin prepolymers , Urethane prepolymer, polyethylene glycol, and polyvinyl alcohol), and

(ii) membrane restraint of cells by immobilization behind the barrier. Barriers may be droplets of a cell-water suspension emulsified in an organic solvent or semi-permeable membrane.

In the process of the present invention, the cell growth (or rate of cell growth)

(i) the amount or rate of new culture medium or nutrient to be introduced into the culture vessel, and / or

(ii) the amount or rate of liquid culture medium comprising the solvent (s) removed from the culture vessel.

The purpose of the process of the present invention is to maintain cell growth and optimize cell density in culture vessels to maximize sugar utilization and solvent production.

Lane growth can be detected by monitoring the cell growth of Clostridia in the culture container. Optimal growth can then be pursued or obtained by introducing a new culture medium or nutrient into the culture vessel and / or by removing some liquid culture medium containing inhibitory solvent.

Thus, preferably, the growth of cells in the culture vessel is maintained at an optimal level.

In other embodiments, when the secondary growth of cells is detected, fresh media or nutrients are introduced into the culture vessel, and / or the liquid medium comprising the solvent is removed from the culture vessel.

In still other embodiments, the monitoring level of cell growth is compared to a reference level, and if the monitored level is below a reference level, a new medium or nutrient is introduced into the culture vessel and / or a liquid medium comprising solvent And removed from the culture container.

The present invention also provides a system for producing a solvent, wherein a system (e.g., as shown in Figure 1)

(i) a culture vessel for culturing the solvent-producing clostridium in a liquid culture medium;

(ii) a monitoring device for monitoring the growth of cells in the culture vessel;

(ii) a cell seeder in fluid communication with the culture vessel and configured to provide clostridium cells to a culture vessel, and / or (iiib) a culture vessel in fluid communication with the culture vessel, A cell separator configured to separate the stridium cells from the liquid culture medium and configured to return the separated cells to the culture vessel;

(iv) a liquid culture medium that is configured to receive the liquid culture medium from cells optionally in fluid communication with the culture vessel and optionally removed by a cell separator configured to separate one or more solvents from the liquid culture medium, A solvent remover configured to return to the culture vessel;

(v) one or more flow regulators in fluid communication with the culture vessel to control the rate or amount of fresh culture medium introduced into the culture vessel and / or the rate or amount of liquid culture medium delivered to the cell separator; And

(vi) one or more controllers for controlling said one or more flow conditioners according to an input received from the monitoring device.

In addition, the system may, optionally,

(vii) a distillation tube through which the culture medium is guided at the end of the process to remove the bulk of the water from the solvent, for example, by azeotropic distillation;

(viii) at least one rectifying column for purifying the solvent to the desired product specification;

(ix) a stillage treatment section for reducing COD / BOD (chemical oxygen demand / biochemical oxygen demand), optionally also for returning energy from a final culture medium with anaerobic fermentation or the like; And

(x) one or more reservoirs of sugar solution and / or nutrients

≪ / RTI >

In some embodiments of the present invention, the culture vessel comprises single-phase solvent-produced Clostridia in a liquid culture medium.

The system also includes a container adapted to contain or contain an alkali; And / or a container adapted to contain or contain a fresh culture medium (nutrient). This latter container is in fluid communication with the first culture vessel.

The means for liquid communication preferably comprises a pipe.

The system may also include one or more additional solvent removers (e.g., one or more rectification columns, one or more distillation tubes, etc.) configured to remove the solvent from the liquid culture medium in fluid communication with the culture vessel and removed from the culture vessel have.

All of the features of the present invention described above in connection with the process of the present invention apply equally to the system described above with only minor modifications to the required portions.

Figure 1 shows an example of an integrated batch feed process concept of the present invention.
Figure 2 is a batch fermentation profile for abnormal solvent-forming Clostridium. The graph shows that growth is slow and solvent production begins when the cells enter the stationary state. The fermentation time is considerably longer. The final solvent optimum concentration is low, and thus the solvent productivity over the entire fermentation is low (Example 1).
Figure 3 is a batch fermentation profile for single-phase solvent-forming clostridium. The graph shows that solvent production occurs during growth, slowing growth, and slowing down when the cells enter a stationary state. The fermentation time is reduced, and the solvent productivity over the entire fermentation is higher than that of Example 1 (Example 2).
Figure 4 is a batch fermentation profile for single-phase solvent-producing clostridium. The graph shows that when the sugar is consumed, the solvent production is stopped and the sugar is used during solvent production. Fermentation time is fairly short (Example 3).
Figure 5 relates to batch-fed fermentation for single-phase solvent-producing clostridium. Gas stripping is used to remove the solvent and the sugar feed is molasses. The graph shows the accumulation of solvent during the fermentation process (the sum of the solvent is captured externally and the remainder in the fermenter). The resulting final solvent titre concentration is much higher than that obtained in the conventional batch fermentation, Example 3 (Example 4).
Figure 6 relates batch-fed fermentation for single-phase solvent-forming clostridium. Gas stripping is used to remove the solvent and the sugar feed is molasses. The graph shows the correlation between solvent productivity and sugar absorption, and the peak solvent productivity was extended by adding additional sugars during fermentation and by removing the solvent in solvent production (Example 4).
Figure 7 relates to batch-fed fermentation for single-phase solvent-producing clostridium. Gas stripping is used to remove the solvent and the sugar feed is molasses. The graph shows a direct correlation between cell growth and density (measured by optical density) and solvent productivity (Example 4).
Figure 8 relates to batch-fed fermentation for single-phase solvent-producing clostridium. Gas stripping was used to remove the solvent and the saccharide feed was glucose (Example 5). The graph shows the accumulation of solvent during the fermentation process.
Figure 9 relates to a pilot-scale batch-fed fermentation for single-phase solvent-producing Clostridium. The process includes a membrane filtration unit (cell separator) that removes cells from the fermentation broth outside the culture vessel. The cells are recirculated back into the culture vessel and the culture broth removed from the cells is fed to a distillation column (solvent remover) which separates the solvent from the liquid culture medium. The distillate is returned to the culture vessel. The saccharide feedstock is glucose. The graph shows that there is a direct correlation between sugar absorption and solvent production, which can sustain high solvent productivity for more than 40 hours (Example 6).
Figure 10 relates to pilot-scale batch-fed fermentation for single-phase solvent-producing Clostridium. The process includes a membrane filtration unit (cell separator) that removes cells from the fermentation broth outside the culture vessel. The cells are recirculated back into the culture vessel and the culture broth removed from the cells is fed to a distillation column (solvent remover) which separates the solvent from the liquid culture medium. The distillate is returned to the culture vessel. The sugar feed is corn mash. The graph shows that industrial corn-based feedstocks can be used to maintain solids absorption and solvent productivity for more than 40 hours (Example 7).
Figure 11 relates to pilot-scale batch-fed fermentation for single-phase solvent-producing Clostridium. The process includes a membrane filtration unit (cell separator) that removes cells from the fermentation broth outside the culture vessel. The cells are recirculated back into the culture vessel and the culture broth removed from the cells is fed to a distillation column (solvent remover) which separates the solvent from the liquid culture medium. The distillate is returned to the culture vessel. The sugar feed is molasses. The graph shows the correlation between glucose uptake and solvent productivity during fermentation (Example 8).
Figure 12 relates to pilot-scale batch-fed fermentation for single-phase solvent-producing clostridium. The process includes a membrane filtration unit (cell separator) that removes cells from the fermentation broth outside the culture vessel. The cells are recirculated back into the culture vessel, and the culture broth removed from the cells is fed to a distillation column (solvent remover) which separates the solvent from the liquid culture medium. The distillate is returned to the culture vessel. The sugar feed is molasses. The graph shows the correlation between gas production and solvent productivity during fermentation (Example 8).

Example

Unless otherwise noted, the present invention is further illustrated in the following examples in which parts and percentages are by weight and degrees Celsius. It should be understood that these embodiments are illustrative of preferred embodiments of the present invention, but are for illustrative purposes only. It will be apparent to those of ordinary skill in the art from the foregoing description and these embodiments that various modifications and variations of the present invention may be made without departing from the spirit and scope of the invention, Can be performed. Accordingly, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also within the scope of the claims.

In laboratory scale (1L) embodiments, gas-stripping is used to maintain a low level of solvent in the main fermenter. However, in pilot scale (140 L) embodiments (and preferred commercial methods) solvent removal is performed using atmospheric distillation with nutrients and water recirculation to the main fermenter.

Comparative Example  Batch fermentation with 1: or more solvent-generated clostridium

purpose

Demonstrating the anomalous batch fermentation profiles obtained using traditional over-solvent-producing Clostridia.

Materials and methods

Bacterial strain

Solvent-generated Clostridium Beijerring strains BAS in a standard anaerobic culture medium, such as enhanced Clostridium medium (RCM as in Table 1), in 100 mL serum bottles under anaerobic conditions for 16-18 hours at 32 1 C / B2 (CSC / NCP collection 15/06/1945) were cultured.

g < RTI ID = 0.0 > L- ¹ < Yeast extract 3.0 Experimental Shim -Lemco Powder 10.0 peptone 10.0 Glucose 5.0 Soluble starch 1.0 Sodium chloride 5.0 Sodium acetate 3.0 Cysteine hydrochloride 0.5 Agar 0.5 * pH 6.8 ± 0.2

 * Adjusted to meet performance standards, if necessary, and sterilized by autoclaving at 121 ° C

Fermentation medium

Molasses was the primary carbon source for the fermentation medium. The experiment was started at an initial volume of 0.95 L at a molar sugar concentration of 60 g · L ^-1 in a fermentation vessel, and then subjected to 5 g · L ^-1 corn silicate, 5 g · L ^-1 CaCO ₃ and 2 g · L ^-1 (NH ₄ ) it was supplemented with a ₂ SO _4. The initial pH was adjusted to 6.7 ± 0.2 by 20% (w / v) NaOH and the pH was not controlled. All mineral salts were laboratory grade (Fisher Scientific).

Culture conditions

Fermentation was carried out in a 1 L fermenter with an initial working volume of 0.95 L. The fermenter was equipped with a gas ejector, a stirrer, a sampling port, pH and temperature probes. Fermentation was initiated by inoculation with a final volume (1 L) of 1% v / v seed culture. The temperature was maintained at 32 ± 3 ° C.

Analysis of cell growth, products, and substrates

Growth was monitored at 600 nm using a Jenway 6300 spectrophotometer with a 1 cm light path cuvette. The cultures were diluted as needed so that the absorbance did not exceed 0.6 units.

The concentrations of acetate, butyrate, ethanol, acetone, and n-butanol were determined by applying the supernatant from the centrifuged fermentation samples to gas chromatography on an Agilent gas chromatography system with a network sampler. The instrument was equipped with a capillary column (Agilent 19091F115E HP-FFAP) with a column temperature ramp from 80 ° C to 200 ° C. The carrier gas had N ₂ at a flow rate of 0.8 mL min ^-1 to 1.3 mL min ^-1 . The hydrogen flow rate was 50 mL · min ^-1 , the air flow rate was 400 mL · min ^-1 , and the makeup flow rate (N ₂ ) was 30 mL · min ^-1 , the FID detector temperature (300 ° C.) was handled. Isobutanol (99.5%) and isobutyric acid (99.5%) were used as internal standards, each prepared at a concentration of 10 g · L ^-1 in HPLC grade water.

The sugar content of the fermentation samples was determined by high pressure liquid chromatography using Dionex HPLC equipped with ASI00 autosampler and Shodex RI 101 refractive index and US detector. The separation column was a Phenomonex Rezex-Pb ^{+ 2} operating at 85 ℃. HPLC grade water was used as mobile phase at a flow rate of 0.6 mL / min. The sample injection volume was 10 μl. Calibration curves are generated by incorporating peak areas from chromatograms generated from sucrose, maltose, glucose, or fructose (depending on the specific source used) at concentrations of 1, 5, 10, 15 and 20 g L ^-1 Respectively.

result

The results are shown in FIG. The graph shows that growth begins to slow and solvent production begins when the cells enter the stationary phase. The fermentation time is considerably longer (55 hours). The final solvent optimum concentration is low, and therefore, the solvent productivity over the entire fermentation is low.

Comparative Example  2: Production of Single Phase Solvent Batch Fermentation by Clostridium

purpose

Phase batch fermentation profile obtained using single-phase solvent-forming clostridium.

Materials and methods

Bacterial strain

Solvent-producing clostridium saccharoprotein acetonucleoside strains N1-4 (HMT) were grown in 100 ml serum bottles under anaerobic conditions for 16 to 18 hours at 32 1 C and in enhanced clostridial medium The same RCM) were cultured in a standard anaerobic culture medium.

Fermentation medium

As in Example 1, molasses was the primary carbon source for the fermentation medium. The experiment was started at an initial volume of 0.95 L at a molar sugar concentration of 60 g · L ^-1 in a fermentation vessel, and then subjected to 5 g · L ^-1 corn silicate, 5 g · L ^-1 CaCO ₃ and 2 g · L ^-1 (NH ₄ ) it was supplemented with a ₂ SO _4. The initial pH was adjusted to 6.7 ± 0.2 by 20% (w / v) NaOH and the pH was not controlled.

Culture conditions

Similar to Example 1, fermentation was performed in a 1 L fermenter with an initial working volume of 0.95 L. The fermenter was equipped with a gas ejector, a stirrer, a sampling port, pH and temperature sensors. Fermentation was initiated by inoculation with a final volume (1 L) of 1% v / v seed culture. The temperature was maintained at 32 ± 3 ° C.

Analysis of products and substrates

As described in the first embodiment.

result

The results are shown in FIG. The graph shows that as the growth slows and the cells enter the stationary phase, solvent production occurs and slows during growth. The fermentation time was reduced (45 hours) and the solvent productivity over the entire fermentation was higher than in Example 1.

Comparative Example  3: Single phase solvent production Batch fermentation by Clostridium

purpose

Single phase solvent production This demonstrates the relationship between sugar utilization and ABE production for batch fermentation using Clostridium.

Materials and methods

Bacterial strain

Solvent-producing clostridium saccharopheyl acetonucleoside strains N1-4 (HMT) were cultured as described in Example 2.

Fermentation medium

Similar to Example 2, molasses was the primary carbon source for the fermentation medium. After the fermentation vessel began to experiment in the initial volume of 0.95L with molasses sugar concentration of 55g · L ^-1 of 2.5g · L ^-1 yeast extract, 2.5g · L ^-1 tryptone, 0.025g · L ^-1 of FeSO ₄ , and (NH ₄ ) ₂ SO ₄ of 0.5 g · L ^-1 .

Culture conditions

Similar to Example 2, fermentation was carried out in a 1 L fermenter with an initial working volume of 0.8 L. The fermenter was equipped with a gas ejector, a stirrer, a sampling port, pH and temperature sensors. Fermentation was initiated by inoculation with a final volume (1 L) of 7.5% v / v seed culture. The temperature was maintained at 32 ± 3 ° C. The initial pH was adjusted to 6 to 6.5 using 20% w / v NaOH and the pH set point was maintained at 5.0 to 5.5, as needed during fermentation. All mineral salts were laboratory grade (Fisher Scientific).

Analysis of products and substrates

As described in the first embodiment.

result

The result is shown in FIG. The graph shows that sugar is used during solvent production and solvent production is discontinued when sugar is consumed. Fermentation time is fairly short (44 hours).

Example  4: Fermentation batch feed fermentation

purpose

By using a low concentration of solvent in the broth (using gas stripping), we focus on the production of cell populations growing exponentially at maximum sugar uptake for extended periods of time compared to traditional batch fermentations, or close to maximum sugar uptake Demonstrate a method of culturing single-phase solvent-producing clostridium at high production rates using a tailored batch supply system.

Materials and methods

Bacterial strain

Solvent-producing clostridium saccharopheyl acetonucleoside strains N1-4 (HMT) were cultured as described in Example 2.

Fermentation medium

Molasses was the primary carbon source for the fermentation medium. After starting the experiment in an initial volume of 1.5 L at a sugar concentration of 50 g · L ^-1 in a fermentation vessel, a 2.5 g L ^-1 yeast extract, a 2.5 g L ^-1 tryptone, a 0.5 g L ^-1 NH ₄ ) ₂ SO ₄ , 0.025 g · L ^-1 FeSO ₄ , and 3 g · L ^-1 CaCO ₃ were supplemented. If necessary, the pH was adjusted to pH 6.5 with 20% w / v NaOH before sterilization. All mineral salts were laboratory grade (Fisher Scientific).

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3.8 g L ^-1 of yeast extract, 3.8 g L ^-1 of tryptone, 0.8 g L ^-1 of (NH ₄ ) ₂ SO ₄ , 3 g L ^-1 of CaCO ₃ and 0.05 g L ^-1 Of FeSO ₄ , together with 500 mL of a 350 g L < ^-1 > glucose solution,

Culture conditions

The fermentation was carried out in a fermenter equipped with a gas ejector, a stirrer, a sampling port, pH and temperature sensors. Fermentation was initiated by inoculation with 7.5% v / v seed culture.

Immediately after the inoculation, the feed container 1L was connected to the fermenter (Fig. 1). When the butanol concentration reached 1 g / L after 6 hours, product stripping via nitrogen stripping was initiated at a gas flow rate of ₂ vvm N ₂ . Batch feeding mode operation began approximately 15 hours after inoculation. Once the sugar concentration in the vessel was reduced to less than the set value (26 g · L ^-1 ), the media reservoir pump was connected and operated. The media store provided new sugars and nutrients. Feed pumps operated automatically and intermittently according to glucose uptake and culture growth. The temperature was maintained at 32 3 C and the control system was adjusted to maintain a minimum of agitation (50-70 rpm). The pH was not controlled.

Analysis of products and substrates

As described in the first embodiment.

result

Figure 5 shows the accumulation of solvent during the fermentation process (the sum of the trapped solvent remaining outside and the rest remaining in the fermenter). The final optimum solvent concentration obtained is much higher than that obtained in the conventional batch fermentation, that is, Example 3.

Figure 6 shows the correlation between solvent productivity and glucose uptake and shows that peak solvent productivity has been expanded by adding additional sugars during fermentation and removing the solvent as the solvent is produced (the plotted data is the accumulation average).

Figure 7 shows a direct correlation between cell growth and density (as determined by optical density) and solvent productivity (the plotted data are cumulative averages).

It has been found that fermentation controlled solvent concentration values have a significant effect on growth rate and eventually on glucose uptake and solvent production rate. High solvent production rates were maintained over an extended period of time throughout the feed phase and the cultures were growing at or near maximum growth rate. The fermentation lasted for 164 hours, of which 53.5 hours were for feed. The productivity of the solvent was 0.90 g / L / h during the feeding period, 0.79 g / L / h during the total fermentation period, and the sugar uptake rate was 2.9 and 2.7 g / L / h over the feeding period and the total fermentation period, Were 0.32 and 0.30 g solvent * / g sugar used, respectively, throughout the feed and total fermentation periods (* Note: 'Solvent' data are based on the collected solvent and a significant portion of the produced solvent was not captured, So that the actual value of the solvent produced will be higher).

Example 5: Glucose  Batch fed fermentation

purpose

Demonstrating that the batch delivery system described above (Example 4) for culturing single-phase solvent-producing clostridium at high production rates effectively functions using other sugars, for example crystallized glucose.

Materials and methods

Bacterial strain

Solvent-producing clostridium saccharopheyl acetonucleoside strains N1-4 (HMT) were cultured as described in Example 2.

Fermentation medium

Crystallized glucose was the major carbon source for the fermentation medium. After the fermentation vessel began to experiment in the initial volume of 1.55L to a sugar concentration of 53g · L ^-1 of 2.5g · Trip ton of L ^-1, 2.5g · L ^-1 yeast extract, 0.5g · L ^-1 of ( NH ₄ ) ₂ SO ₄ , 0.025 g · L ^-1 FeSO ₄ , and 3 g · L ^-1 CaCO ₃ were supplemented.

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3.8 g L ^-1 yeast extract, 3.8 g L ^-1 tryptone, 0.8 g L ^-1 (NH ₄ ) ₂ SO ₄ , 0.05 g L ^-1 FeSO ₄ And CaCO ₃ nutrients, an additional feed of 650 mL of a glucose solution at a concentration of 492 g · L ^-1

The sugar solution and nutrient-based media were batch processed and individually sterilized.

Culture conditions

The fermentation was carried out in a 2L fermenter. The fermenter was equipped with a gas ejector, a stirrer, a sampling port, pH and temperature sensors. Fermentation was initiated by inoculation with 7.5% v / v seed culture. Inoculation with 7.5% v / v seed culture was repeated every 48 hours.

The feed container 1L was connected to a fermenter. Once the initial sugar concentration had dropped to less than ~ 25 g / L, feed addition (concentrated sugar and nutrient addition) was started and product stripping by gas stripping (≥2 vvm) began. Feed pumps operated automatically and intermittently according to sugar uptake and culture growth. The control system was adjusted to maintain the temperature at 32 1 C and maintain a minimum of agitation (50-70 rpm).

Analysis of products and substrates

As described in the first embodiment.

result

The result is shown in Fig. The graph shows the accumulation of solvent during the fermentation process. Fermentation lasted 216.5 hours, of which 191 hours were for feed. The productivity of the solvent was 0.57 g / L / h during the feeding period, 0.53 g / L / h during the total fermentation period, and the sugar uptake rate was 2.1 and 2.0 g / L / h respectively over the feeding period and the total fermentation period, Were 0.21 and 0.20 g solvent * / g sugar used, respectively, throughout the feed and total fermentation periods (* Note: 'solvent' data are based on the collected solvent and a significant portion of the produced solvent was not captured, So that the actual value of the solvent produced will be higher).

Example  6: Pilot-scale batch fed fermentation of glucose

purpose

This batch feed fermentation process proves that glucose works effectively on a pilot scale using it as a major sugar source. The system of this embodiment includes a membrane filtration unit (cell separator) that removes cells from the fermentation broth outside the culture vessel. The cells are recirculated back into the culture vessel and the culture broth minus the cells is supplied to a distillation column (solvent remover) which separates the solvent from the liquid culture medium. The distillate is returned to the culture vessel (see Fig. 1).

Bacterial strain

Solvent-producing clostridium saccharoprotein acetonucleoside strains N1-4 (HMT) were cultured in 38 g · L ¹ Difco RCM medium at 32 ± 1 ° C for 16 to 18 hours in anoxic condition. L of glucose, 40 g / L of glucose, 5 g / L of yeast extract, 5 g / L of meat peptone, 2 g / L of (NH ₄ ) ₂ SO ₄ , 0.05 g / L of FeSO ₄ , L and sodium triacetate trihydrate (3 g / L) were inoculated with 5 L seed carboy (carboy). This was incubated for 5.25 hours to generate 5 L of seed.

Fermentation medium

Glucose was used as the primary carbon source for the fermentation broth. 150 L of the following medium was prepared. L of peptone, 2 g / L of (NH ₄ ) ₂ SO ₄ , 0.05 g / L of FeSO ₄ , 0.1% v / v of corn oil, 2343) 0.03 g / L.

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50 L concentrated feed containing 450 g / L glucose.

The 16 L nutrient addition solution contains 58 g / L yeast extract, 58 g / L meat peptone, 17 g / L (NH ₄ ) ₂ SO ₄ , 0.4 g / L FeSO ₄ and 0.4% v / v corn oil oil.

The sugar and nutrient-based media were batch processed and individually sterilized.

Culture conditions

Initiation of fermentation in a 140 L fermentor, initially 105 L of fermentation medium was combined with 5 L of seed.

The temperature was maintained at 30.5 ± 0.3 ° C. The pH dropped rapidly after inoculation until pH control started at pH 5.3 using 10-25% w / v NaOH.

Analysis of products and substrates

As described in the first embodiment.

result

The result is shown in FIG. The plotted values are five point averages. The graph shows that there is a direct correlation between glucose uptake and solvent production, which can maintain high solvent productivity for about 50 hours. During the entire 102.5 hours fermentation, the average productivity was 0.68 g / L / h, the productivity during the batch feed phase of 52 hours was 1.11 g / L / h and the sugar consumption over the entire 102.5 hours fermentation was 1.91 g / L / h , The consumption of sugar per batch feed phase of 52 hours was 2.77 g / L / h, the solvent production based on the final volume of 120 L was 70 g / L, the ABE yield to consumed sugar was 35.7%, and the butanol ratio 76%.

Example  7: Refined corn Hourly  Pilot scale batch feed fermentation

purpose

This batch feed fermentation process proves to work effectively on a pilot scale using refined corn mash as a state party. The system of this embodiment includes a membrane filtration unit (cell separator) that removes cells from the fermentation broth outside the culture vessel. The cells are recirculated back into the culture vessel, and some of the culture broth minus the cells are fed to a distillation column (solvent remover) which separates the solvent from the liquid culture medium. The distillate is returned to the culture vessel (see Fig. 1).

Bacterial strain

Solvent-producing clostridium saccharoprotein acetonucleoside strains N1-4 (HMT) were cultured in 38 g · L ¹ Difco RCM medium at 32 ± 1 ° C for 16 to 18 hours in anoxic condition. Using 150 mL of this, 150 μL of a purified refined corn meal medium / seed (refined corn silicate, yeast extract 5 g / L, mitepectone 5 g / L, equivalent to 40 g / L, (NH ₄ ) ₂ SO ₄ 2 g / L , 0.05 g / L FeSO ₄ and ₃ g / L sodium acetate trihydrate). This was incubated for 5.25 hours to generate 5 L of seed.

Fermentation medium

The refined corn meal was used as the main carbon source for the fermentation medium. 105 L of the following medium was prepared. 2.5 g / L of yeast extract, 2 g / L of (NH ₄ ) ₂ SO ₄ , and 0.05 g / L of FeSO ₄ ), equivalent to 60 g / L of purified corn silicate.

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A 75 L concentrate feed containing purified corn mash equal to 270 g / L.

The 8 L nutrient addition solution contains 95 g / L yeast extract, 65 g / L (NH ₄ ) ₂ SO ₄ , and 1.7 g / L FeSO ₄ .

The sugar and nutrient-based media were batch processed and individually sterilized.

Culture conditions

Initiation of fermentation in a 140 L fermenter, initially 100 L of fermentation medium was combined with 5 L of seed.

The temperature was maintained at 30.5 ± 0.3 ° C. The pH dropped rapidly after inoculation until pH control started at pH 5.3 using 10-25% w / v NaOH.

Analysis of products and substrates

As described in the first embodiment.

result

The results are shown in FIG. The plotted values are five point averages. The graph shows that industrial corn-based feedstocks can be used to maintain solids absorption and solvent productivity for about 50 hours.

The average productivity over the 110 hour fermentation cycle (97.75 hours fermentation + CIP (cleaning at the site) + reciprocating time) was 0.87 g / L / h and productivity during the batch delivery phase of 52 hours was 1.51 g / L / h, the sugar consumption during the 110 hour fermentation cycle was 2.84 g / L / h, the sugar consumption during the 52 hour batch feed phase was 5.25 g / L / h and the solvent production based on the final volume of 105 L was 96 g / L, and the ABE yield was 30.6% and the butanol ratio was 61.5% for the saccharide consumed.

Example  8: Pilot-scale batch batch feed fermentation using gas production monitoring

purpose

This batch feed fermentation process demonstrates that molasses is used effectively as a primary feedstock and functions effectively on a pilot scale. The system of this embodiment includes a membrane filtration unit (cell separator) that removes cells from the fermentation broth outside the culture vessel. The cells are recirculated back into the culture vessel and the culture broth minus the cells is supplied to a distillation column (solvent remover) which separates the solvent from the liquid culture medium. The distillate is returned to the culture vessel (see Fig. 1). It also monitors the gases from fermentation.

Bacterial strain

Solvent-producing clostridium saccharoprotein acetonucleoside strains N1-4 (HMT) were cultured in 38 g · L ¹ Difco RCM medium at 32 ± 1 ° C for 16 to 18 hours in anoxic condition. L of molasses, 5 g / L of meat yeast extract, 5 g / L of meat peptone, 2 g / L of (NH ₄ ) ₂ SO ₄ , 0.05 g / L of FeSO ₄ , L and sodium triacetate trihydrate 3 g / L). This was incubated for 5.25 hours to generate 5 L of seed.

Fermentation medium

The sugarcane molasses was used as the main carbon source for the fermentation medium. 105 L of the following medium was prepared. L of peptone, 2 g / L of (NH ₄ ) ₂ SO ₄ , 0.05 g / L of FeSO ₄ , 0.1% of corn oil, 50 g / L of yeast extract, v / v and defoamer (Suppressor 2343) 0.03 g / L).

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A 50 L concentrate feed containing sugar cane molasses equivalent to 350 g / L.

16 L of the nutrient addition solution contains 34 g / L of yeast extract, 34 g / L of meatpeptone, 20 g / L of (NH ₄ ) ₂ SO ₄ , 0.5 g / L of FeSO ₄ and 0.6% of corn oil.

The sugar and nutrient-based media were batch processed and individually sterilized.

Culture conditions

Initiation of fermentation in a 140 L fermentor, initially 105 L of fermentation medium was combined with 5 L of seed.

The temperature was maintained at 30.5 ± 0.3 ° C. No calcium carbonate or other buffer was added. The pH dropped rapidly after inoculation until pH control started at pH 5.3 using 10-25% w / v NaOH.

The butanol concentration of the culture vessel was kept below 6 g / l and the sugar concentration of the culture vessel was maintained at 20 g / L to 30 g / L during feeding.

Analysis of products and substrates

As described in the first embodiment.

result

The results are shown in FIG. 11 and FIG. The plotted values are five point averages. Figure 11 shows the correlation between glucose uptake and solvent productivity during fermentation and indicates that high molar absorption and solvent productivity can be maintained for extended periods using industrial molasses-based feedstocks. Figure 12 shows the correlation between gas production and solvent productivity during fermentation.

The average productivity during the 67 hour fermentation was 0.60 g / L / h, the productivity during the 30 hour batch feed phase was 0.95 g / L / h and the sugar consumption during the 67 hour fermentation was 1.67 g / L / h , The sugar consumption during the 30 hour batch feed phase was 2.74 g / L / h, the ABE yield for the consumed sugar was 35.8%, and the butanol ratio was 75%.

Claims (20)

(a) culturing single-phase solvent-producing clostridia cells in a liquid culture medium in a culture vessel;
(b) monitoring cell growth of the single-phase solvent-produced Clostridia cells in the culture vessel;
(c) introducing new culture medium and / or nutrients continuously or semi-continuously into the culture vessel;
(d) continuously or semi-continuously removing the stream or part of the liquid culture medium containing solvent (s) from the culture vessel and delivering the liquid culture medium to a solvent remover;
(e) maintaining or increasing the cell density in the culture vessel,
These cells are derived from monoclonal clostridial sacaropteryl acetonickum ,
The cell growth of the culture vessel is carried out,
(i) the amount or rate of new culture medium or nutrient to be introduced into the culture vessel, and / or
(ii) controlling and / or controlling the amount or rate of the liquid culture medium comprising the solvent (s) removed from the culture vessel,
Single-stage fed-batch or continuous process for solvent production.
The method according to claim 1,
The step (e)
e) increasing the cell density in the culture vessel,
The cell growth of the culture vessel is carried out,
(i) the amount or rate of new culture medium or nutrient to be introduced into the culture vessel, and / or
(ii) controlling the amount or rate of the liquid culture medium comprising the solvent (s) removed from the culture vessel,
Wherein said process maintains cell growth, optimizes cell density in said culture vessel, and maximizes sugar utilization and solvent production.
3. The method according to claim 1 or 2,
The monoclinic clostridium may be,
(a) naturally mark concurrent growth and solvent production during the main growth phase of batch fermentation,
(b) chemically modified to produce a solvent during growth, or
(c) a genetically modified process to produce a solvent during growth.
The method of claim 3,
Wherein said clostridium is a monoclonal clostridial saccharoprotein acetonucleic N1 strain, preferably N1-4 (HMT) or N1-504, or a variant or derivative thereof.
5. The method according to any one of claims 1 to 4,
The step (b)
(i) monitoring the production of one or more gases;
(ii) monitoring the production of one or more acids;
(iii) monitoring the pH of the culture medium;
(iv) monitoring the usage per party;
(v) monitoring the cell density; And
(vi) monitoring the production of one or more solvents.
6. The method according to any one of claims 1 to 5,
The monitoring level of cell growth is compared with a reference level and, if the monitored level is lower than the reference level, a new medium or nutrient is introduced into the culture vessel and / or a liquid medium containing solvent is removed from the culture vessel. fair.
7. The method according to any one of claims 1 to 6,
The step (d)
Continuously or semicontinuously removing a stream or part of the liquid culture medium from the culture vessel and delivering the stream / portion to the solvent remover via a cell separator,
(i) the cells are removed from the liquid culture medium in a cell separator, the cells are returned to the culture vessel (optionally via a cell seeder), and / or
(ii) the solvent is removed from the liquid culture medium in a solvent remover, and the remaining liquid culture medium is returned to the culture vessel.
8. The method according to any one of claims 1 to 7,
Wherein the solvent extraction is carried out continuously.
9. The method according to any one of claims 1 to 8,
Wherein the liquid culture medium comprising the solvent (s) is removed from the culture vessel at a rate to maintain the solvent concentration of the liquid culture below a desired solvent concentration point.
10. The method of claim 9,
Wherein the concentration point is a toxicity threshold for the solvent-produced Clostridium.
10. The method of claim 9,
Wherein the defined solvent concentration point is 8-10 g butanol / L.
12. The method according to any one of claims 1 to 11,
Wherein the butanol concentration of the culture vessel does not exceed 10 g / L.
13. The method according to any one of claims 1 to 12,
The cell density,
(i) recycling the cells removed from the liquid culture medium containing the solvent (s) back to the culture vessel; And / or
(ii) continuously or semicontinuously feeding cells from a cell cider into a culture vessel; And / or
(iii) the cell is maintained or increased in the culture vessel by securing some or all of the cells in the culture vessel.
14. The method according to any one of claims 1 to 13,
Wherein the sugar content of said culture vessel is maintained at 5-30 g / L.
15. The method according to any one of claims 1 to 14,
Wherein said cells are maintained under redox conditions suitable for optimal growth to produce a solvent.
16. The method according to any one of claims 1 to 15,
Wherein the single-phase solvent-produced Clostridia cells are cultured in a liquid culture medium of the culture vessel without prior anaerobic purging.
(i) a culture vessel for culturing the solvent-producing clostridium in a liquid culture medium;
(ii) a monitoring device for monitoring the growth of cells in the culture vessel;
(ii) a cell seeder in fluid communication with the culture vessel and configured to provide clostridium cells to a culture vessel, and / or (iiib) a culture vessel in fluid communication with the culture vessel, A cell separator configured to separate the stridium cells from the liquid culture medium and configured to return the separated cells to the culture vessel;
(iv) a liquid culture medium that is configured to receive the liquid culture medium from cells optionally in fluid communication with the culture vessel and optionally removed by a cell separator configured to separate one or more solvents from the liquid culture medium, A solvent remover configured to return to the culture vessel;
(v) one or more flow regulators in fluid communication with the culture vessel to control the rate or amount of fresh culture medium introduced into the culture vessel and / or the rate or amount of liquid culture medium delivered to the cell separator; And
(vi) one or more controllers for controlling said one or more flow conditioners in accordance with an input received from a monitoring device
System for solvent production.
18. The method of claim 17,
The system comprises:
(vii) a distillation tube through which the culture medium is guided at the end of the process to remove the bulk of the water from the solvent;
(viii) at least one rectifying column for purifying the solvent to the desired product specification;
(ix) a stillage treatment section for reducing COD / BOD, optionally also for returning energy from the final culture medium; And
(x) at least one of at least one reservoir of sugar solution and / or nutrient in fluid communication with the culture vessel.
The method according to claim 17 or 18,
Wherein the culture vessel comprises single-phase solvent-producing Clostridia cells in a liquid culture medium.
20. The method of claim 19,
Wherein the clostridium is a single phase clostridial saccharoprotein acetonucleic N1 strain, preferably N1-4 (HMT) or N1-504, or a variant or derivative thereof.

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